The common theme underlying the questions being addressed in this proposal is how gene expression is controlled by cis-regulatory modules (CRMs) and the transcription factors that they bind during animal development. Answers to this question are becoming increasingly important in the emerging era of Personalized Medicine, since we now have the ability to sequence thousands of genomes and to identify changes in DNA sequences that correlate with diseases ranging from epilepsy to obesity. However, when changes in DNA sequence map to the non-protein coding portion of the genome, we are nearly helpless in interpreting these changes. This project will help bridge this gap in our knowledge by establishing methods to assess how the Hox family of transcription factors function in vivo. In the next several years, one goal is to extend what has been learned about Hox transcription factor specificity to a more in vivo level, in particular, to break the sequence code that allows these TFs to select biologically relevant binding sites in vivo. A second major goal is to understand at a deeper level how multiple transcription factors and CRMs coordinate with each other to regulate genes in the right cells and at the correct developmental time. For this last set of experiments, the focus is on how the proximo-distal axis of the Drosophila leg is established during development.